Chemotaxis proteins and transducers for aerotaxis in Pseudomonas aeruginosa

It was previously shown that the chemotaxis gene cluster 1 (cheYZABW) was required for chemotaxis. In this study, the involvement of the same cluster in aerotaxis is described and two transducer genes for aerotaxis are identified. Aerotaxis assays of a number of deletion–insertion mutants of Pseudomonas aeruginosa PAO1 revealed that the chemotaxis gene cluster 1 and cheR are required for aerotaxis. Mutant strains which contained deletions in the methyl-accepting chemotaxis protein-like genes tlpC and tlpG showed decreased aerotaxis. A double mutant deficient in tlpC and tlpG was negative for aerotaxis. TlpC has 45 0x1.e0b6p-891mino acid identity with the Escherichia coli aerotactic transducer Aer. The TlpG protein has a predicted C-terminal segment with 89 0dentity to the highly conserved domain of the E. coli serine chemoreceptor Tsr. A hydropathy plot of TlpG indicated that hydrophobic membrane-spanning regions are missing in TlpG. A PAS motif was found in the N-terminal domains of TlpC and TlpG. On this basis, the tlpC and tlpG genes were renamed aer and aer-2, respectively. No significant homology other than the PAS motif was detected in the N-terminal domains between Aer and Aer-2

A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy

動物由来の成分を含まないより安全な製法でiPS細胞から大量の再生T細胞を培養する方法の開発 --T細胞を使ったがん免疫療法での利用も--. 京都大学プレスリリース. 2021-01-18.Clinical successes demonstrated by chimeric antigen receptor T-cell immunotherapy have facilitated further development of T-cell immunotherapy against wide variety of diseases. One approach is the development of “off-the-shelf” T-cell sources. Technologies to generate T-cells from pluripotent stem cells (PSCs) may offer platforms to produce “off-the-shelf” and synthetic allogeneic T-cells. However, low differentiation efficiency and poor scalability of current methods may compromise their utilities. Here we show improved differentiation efficiency of T-cells from induced PSCs (iPSCs) derived from an antigen-specific cytotoxic T-cell clone, or from T-cell receptor (TCR)-transduced iPSCs, as starting materials. We additionally describe feeder-free differentiation culture systems that span from iPSC maintenance to T-cell proliferation phases, enabling large-scale regenerated T-cell production. Moreover, simultaneous addition of SDF1α and a p38 inhibitor during T-cell differentiation enhances T-cell commitment. The regenerated T-cells show TCR-dependent functions in vitro and are capable of in vivo anti-tumor activity. This system provides a platform to generate a large number of regenerated T-cells for clinical application and investigate human T-cell differentiation and biology

Identification and Characterization of the Chemotactic Transducer in Pseudomonas aeruginosa PAO1 for Positive Chemotaxis to Trichloroethylene

Pseudomonas aeruginosa PAO1 is repelled by trichloroethylene (TCE), and the methyl-accepting chemotaxis proteins PctA, PctB, and PctC serve as the major chemoreceptors for negative chemotaxis to TCE. In this study, we found that the pctABC triple mutant of P. aeruginosa PAO1 was attracted by TCE. Chemotaxis assays of a set of mutants containing deletions in 26 potential mcp genes revealed that mcpA (PA0180) is the chemoreceptor for positive chemotaxis to TCE. McpA also detects tetrachloroethylene and dichloroethylene isomers as attractants

Natural stone assessment with ground penetrating radar

The involvement of the chemotaxis gene cluster 1 (cheYZABW) and cheR in repellent responses of Pseudomonas aeruginosa to trichloroethylene (TCE) is described and three methyl-accepting chemotaxis proteins (MCPs) for TCE are identified. TCE chemotaxis assays of a number of deletion-insertion mutants of P. aeruginosa PAO1 revealed that the chemotaxis gene cluster 1 and cheR are required for negative chemotaxis to TCE. Mutant strains which contained deletions in pctA, pctB and pctC showed decreased responses to TCE. The pctA, pctB and pctC genes have been reported to encode MCPs for amino acids [K. Taguchi et al., Microbiology, 143, 3223–3229 (2000)]. The pctA mutation more severely impaired chemotactic responses to TCE than did those of pctB and pctC, suggesting that PctA is the major MCP for TCE among the three MCPs. The pctA, pctB and pctC mutant strains showed decreased responses to chloroform and methylthiocyanate. This result demonstrates that PctA, PctB and PctC are also involved in repellent responses to chloroform and methylthiocyanate

New findings of kinase switch in gastrointestinal stromal tumor under imatinib using phosphoproteomic analysis

Despite the initial effectiveness of the imatinib for gastrointestinal stromal tumors (GISTs), patients can’t stop it and eventually relapse by acquired drug resistance. Pathologically, we find some viable cells under the imatinib therapy even in the disappearance region in radiological examination, and these cells are thought to cause these clinical problems. To discover these mechanisms, we investigated the phospho-proteome alternations induced by imatinib treatment using GIST-T1 cell. Because some of tyrosine kinases might be alternatively activated after imatinib exposure, it is quite important to discover increased phosphorylation levels of proteins at proteome scale, and elucidate these proteins with drug resistance. With or without imatinib treatment, extracted proteins from GIST-T1 cells were digested with trypsin and tyrosine-phosphorylated peptides were purified using an immunoaffinity-enriched and labeled with iTRAQ reagent followed by mass spectral analysis. By phosphoproteomic approach, 171 tyrosine phosphorylation sites spanning 134 proteins were identified. Among them, increased tyrosine phosphorylation levels of Fyn(Y420) and focal adhesion kinase (FAK) (Y576) were detected. Increased phosphorylation levels were also confirmed by western blot analysis and knockdown of Fyn or FAK increased the sensitivity toward imatinib. Furthermore, we found that the anti-proliferative effect of imatinib significantly enhanced imatinib resistant GIST-T1 cells which showed constitutive phosphorylation of FAK, treating with FAK selective kinase inhibitor. These results indicate that iTRAQ based quantitative phospho-tyrosine focused phosphoproteomic approaches are powerful method to screen phosphpproteins associated with drug resistance and Fyn or FAK might be potential therapeutic target for the overcoming acquired resistance to imatinib in GIST

Additional file 2: Figure S1. of Identification of definitive serum biomarkers associated with disease activity in primary Sjögren’s syndrome

Functional annotation of differentially expressed proteins in pSS patient sera. Nodes indicate molecular concepts or set of biologically related genes. Name of each node is indicated in black text on the node. The node size represents the proportion of differentially expressed gene symbols in the concepts (e.g., the “chemokine signaling pathway” and “extracellular region” concepts contain 14 and 58 genes, respectively). Length of lines between nodes represents degree of overlap between symbols. Colored lines indicate strength of functional relationship from strong to weak, as follows: red, yellow, green and gray. Green nodes indicate immune response-related molecular concepts, and red nodes indicate platelet-related molecular concepts. (TIF 9752 kb

Additional file 3: Figure S2. of Identification of definitive serum biomarkers associated with disease activity in primary Sjögren’s syndrome

Serum levels of five proteins in pSS, sSS, sicca syndrome and HCs. The five proteins were CXCL13, TNF-R2, CD48, BAFF and PD-L2. Primary SS (pSS), n = 58; secondary SS (sSS), n = 6; other sicca syndrome, n = 13; healthy controls (HCs), n = 38. Differences in quantitative variables were analyzed by the Mann-Whitney U test when comparing two groups and by the Kruskal-Wallis test when comparing multiple groups. *P value <0.05, which was considered significant. (TIF 33972 kb